Na+ dependence of single-channel current and channel density generate saturation of Na+ uptake in A6 cells.
In high-resistance, salt-absorbing epithelia the apical amiloride-sensitive Na+ channel is the key site for regulation of salt and water balance. The saturation of macroscopic Na+ transport through these channels was investigated using A6 epithelial monolayers. The relation between transepithelial Na+ transport (INa) and apical Na+ concentration ([Na+]ap) under short-circuit conditions was studied. Michaelis-Menten analysis of the saturable short-circuit current (Isc) yielded an apparent Michaelis-Menten constant (KmI) of 5 mmol/l and a maximal current (Imax) of 8 microA/cm2. The microscopic parameters underlying INa, namely the single-channel current (i) and the open channel density (No), were investigated by the analysis of current fluctuations induced by the electroneutral amiloride analogue CDPC (6-chloro-3, 5-diaminopyrazine-2-carboxamide). A two-state model analysis yielded the absolute values of i (0.18 +/- 0.01 pA) and No (65.38 +/- 9.57 million channels/cm2 of epithelium) at [Na+]ap = 110 mmol/l containing 50 mumol/l CDPC. Our data indicate that in A6 cells both i and No depend on [Na+]ap. Between 3 and approximately 20 mmol/l the density of conducting pores, No, decreases sharply and behaves again as an almost [Na+]ap-independent parameter at higher [Na+]ap. The single-channel current clearly saturates with an apparent Michaelis-Menten constant, Kmi, of approximately 17 mmol/l. Thus, the [Na+]ap dependence of No as well as the limited transport capacity of the amiloride-sensitive Na+ channel are both responsible for the saturation of INa.
Smets
I
I
Laboratory of Physiology, Limburgs Universitair Centrum, B-3590 Diepenbeek, Belgium.
Zeiske
W
W
Steels
P
P
Van Driessche
W
W
D000584:Amiloride / Q000031:analogs & derivatives / Q000494:pharmacology; D000663:Amphibians; D000818:Animals; D001692:Biological Transport / Q000502:physiology; D002460:Cell Line; D004847:Epithelial Cells / Q000378:metabolism* / Q000502:physiology; D007700:Kinetics; D018408:Patch-Clamp Techniques; D012964:Sodium / Q000378:metabolism*; D026941:Sodium Channel Blockers; D015222:Sodium Channels / Q000378:metabolism / Q000502:physiology*
D026941:Sodium Channel Blockers; D015222:Sodium Channels; C058206:6-chloro-3,5-diaminopyrazine-3-carboxamide; D000584:Amiloride; D012964:Sodium